Significance: Acetogenic bacteria play important roles in the flow of carbon and energy in anaerobic habitats, including the human gastrointestinal tract. The unique autotrophic pathway used by acetogens is termed the acetyl-CoA (or Wood) pathway, and studies have shown that acetogenesis is a fundamental biological process. New and diverse metabolic activities have recently been elucidated for acetogens, including the capacity to derive both carbon and energy from aromatic compounds. These potentials may contribute to the survival and interaction of acetogens in their native habitats. In general, the anaerobic metabolism of aromatic compounds is poorly defined, and the acetogenic metabolism of such substrates is likely coupled to the overall biotransformation of lignin and other aromatic substrates, including aromatic toxic wastes. Abstract of Research Plan: Strictly defined environments for resolution of the metabolic potentials of acetogenic bacteria have been developed, and new enzyme systems central to acetogenesis from aromatic compounds have been discovered. The main goal of this project will be to further resolve these newly realized catalytic activities. The classic acetogen Clostridium thermoaceticum, the human gastrointestinal isolate Peptostreptococcus productus, and other acetogens will be used in this study. Methods will include: enzyme analyses on the flow of carbon and reductant from aromatic compounds; 13C and 14C tracer analyses; high performance liquid chromatographic (HPLC) analyses; high-resolution fast protein liquid chromatography (FPLC) for the purification of enzyme systems; and the use of enzyme-specific antibodies as molecular probes and inhibitors. The specific aims include: (i) Resolving the specificity and diversity of new capacities observed for acetogenesis from aromatic compounds. (ii) Elucidating how carbon and reductant which are derived from aromatic substituent groups are utilized in the acetyl-CoA pathway. (iii) The purification and characterization of new enzyme systems involved in these processes.